Functional Analysis of Cucumis melo CmXTH11 in Regulating Drought Stress Tolerance in Arabidopsis thaliana

Abstract

The CmXTH11 gene, a member of the XTH (xyloglucan endotransglycosylase/hydrolase) family, plays a crucial role in plant responses to environmental stress. In this study, we heterologously expressed the melon gene CmXTH11 in Arabidopsis to generate overexpressing transgenic lines, thereby elucidating the regulatory role of CmXTH11 in water stress tolerance. Using these lines of CmXTH11 (OE1 and OE2) and wild-type (WT) Arabidopsis as experimental materials, we applied water stress treatments (including osmotic stress and soil drought) and rewatering treatments to investigate the response mechanisms of melon CmXTH11 in Arabidopsis under drought stress from a physiological and biochemical perspective. Overexpression of CmXTH11 significantly improved root growth under water stress conditions. The OE lines exhibited longer roots and a higher number of lateral roots compared to WT plants. The enhanced root system contributed to better water uptake and retention. Under osmotic and drought stress, the OE lines showed improved survival rates and less wilting compared to WT plants. Biochemical analyses revealed that CmXTH11 overexpression led to lower levels of malondialdehyde (MDA) and reduced electrolyte leakage, indicating decreased oxidative damage. The activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), were significantly higher in OE lines, suggesting enhanced oxidative stress tolerance. The CmXTH11 gene positively regulates water stress tolerance in Arabidopsis by enhancing root growth, improving water uptake, and reducing oxidative damage. Overexpression of CmXTH11 increases the activities of antioxidant enzymes, thereby mitigating oxidative stress and maintaining cellular integrity under water deficit conditions. These findings suggest that CmXTH11 is a potential candidate for genetic improvement of drought resistance in crops.

Keywords: CmXTH11; antioxidant enzymes; drought tolerance; oxidative stress; water stress.

Figure 1

CmXTH11 protein structure and physicochemical properties analysis. (a) Conserved domains of CmXTH11 protein; (b) signal peptide domain analysis of CmXTH11 protein; (c) transmembrane domain analysis of CmXTH11 protein; (d) SWISS modeling of CmXTH11.

Figure 2

CmXTH11 expression analysis. (a) Tissue-specific expression analysis of CmXTH11; (b) expression levels of CmXTH11 under different hormone treatments. Note: Data in the same column with different letters indicate significant differences (p < 0.05).

Figure 3

Analysis of CmXTH11 amino acid sequences and phylogenetic tree construction. (a) Amino acid sequence alignment: Comparative analysis of the amino acid sequences of CmXTH11 with XTH proteins from six different plant species; (b) phylogenetic tree analysis: Phylogenetic tree showing the evolutionary relationships between CmXTH11 and XTH proteins from various plant species.

Figure 3

Analysis of CmXTH11 amino acid sequences and phylogenetic tree construction. (a) Amino acid sequence alignment: Comparative analysis of the amino acid sequences of CmXTH11 with XTH proteins from six different plant species; (b) phylogenetic tree analysis: Phylogenetic tree showing the evolutionary relationships between CmXTH11 and XTH proteins from various plant species.

Figure 4

Confirmation of transgenic Arabidopsis lines. (a) Construction of the pCAMBIA1303-CaMV35S-CmXTH11 expression vector; (b) expression levels of CmXTH11 in 4-week-old Arabidopsis leaves of WT and two transgenic lines (OE1 and OE2); (c) phenotypic comparison of 8-week-old WT and CmXTH11-OE lines under normal growth conditions. Note: Data in the same column with different letters indicate significant differences (p < 0.05).

Figure 5

Analysis of root growth in WT and OE Arabidopsis lines. (a,b) Phenotypic images of root growth in WT and OE lines grown on 1/2 MS medium; (c) number of lateral roots in WT and OE lines grown on 1/2 MS medium; (d) root length of WT and OE lines grown on 1/2 MS medium. Note: Data in the same column with different letters indicate significant differences (p < 0.05).

Figure 6

The impact of drought stress on seed germination in WT and CmXTH11 overexpressing Arabidopsis. (a) Phenotypic images of root lengths of WT and transgenic lines on 1/2 MS medium containing 0, 100, 200, and 250 mM mannitol after 10 days of cultivation. (b) Phenotypic images of seed germination under the same conditions. (c) Seed germination rates under different mannitol concentrations. (d) Number of green leaves. (e) Root lengths of seeds grown under different mannitol concentrations. (f) Fresh weight of seeds grown under different mannitol concentrations. Note: Data in the same column with different letters indicate significant differences (p < 0.05).

Figure 7

CmXTH11 gene enhances drought resistance in Arabidopsis. (a) Phenotypes of WT and CmXTH11 overexpressing lines (OE1, OE2) under normal growth conditions, after 9 days of drought stress, and after 5 days of rewatering; (b–d): malondialdehyde (MDA) content (b), relative electrolyte conductivity (REC) (c), and relative water content (RWC) (d) in WT and CmXTH11-OE lines under normal growth conditions, after 9 days of drought stress, and after 5 days of rewatering. Note: Data in the same column with different letters indicate significant differences (p < 0.05).

Figure 8

Overexpression of CmXTH11 gene affects ROS accumulation and antioxidant activity in Arabidopsis leaves under drought stress. (a,b) NBT and DAB staining: The staining of leaves with NBT and DAB reveals the accumulation of O²⁻ and H₂O₂ in WT and CmXTH11-OE Arabidopsis plants under normal conditions, drought stress for 9 days, and after 5 days of rewatering. (c,d) ROS content: The quantification of O²⁻ and H₂O₂; (e–g) antioxidant enzyme activity: The activities of POD (e), CAT (f), and SOD (g) measured in leaves of WT and transgenic lines. Note: Data in the same column with different letters indicate significant differences (p < 0.05).

Figure 9

Impact of overexpressing CmXTH11 gene on stomatal characteristics of Arabidopsis under drought stress. (a–c): (a) Stomatal density (b), stomatal aperture, and (c) pore area of WT and CmXTH11 transgenic Arabidopsis leaves under normal growth conditions, after 9 days of drought stress, and after 5 days of rewatering. Note: Data in the same column with different letters indicate significant differences (p < 0.05).